JJC Identification Lab

API-20EAPI-20E
• API-20E is a test kit for the identification of
enteric bacteria (Gram negative bateria that
reside in the intestine)
• easy way to inoculate and read tests relevant
to members of the Family Enterobacteriaceae
and associated organisms.
Procedural Overview
• plastic strip holding twenty mini-test tubes is
inoculated with a saline suspension of a pure
culture of bacteria
• process also rehydrates the dessicated
medium in each tube
• after incubation 18-24 hours at 37°C, the color
reactions are read (some with the aid of added
reagents), and the reactions are converted to a
seven-digit code
• code is fed into the manufacturer’s database & the
computer gives back the identification, usually as
genus and species
• reliability system is very high
• systems like these are in heavy use in many food
and clinical labs
The test consists of the following:
ONPG: galactosidase
ADH: Arginine DeCarboxylase
LDC: Lysine DeCarboxylase
ODC: Ornithine DeCarboxylase
CIT: Citrate utilization
H2S: Hydrogen sulfide production
URE: Urease
TDA: Tryptophan Deaminase
IND: Indole Production
VP: acetoin production (Voges Proskauer)
GEL: Gelatinase
GLU: fermentation / oxidation (Glucose)
MAN: fermentation / oxidation (Mannitol)
INO: fermentation / oxidation (Inositol)
SOR: fermentation / oxidation (Sorbitol)
RHA: fermentation / oxidation (Rhamnose)
SAC: fermentation / oxidation (Saccharose)
MEL: fermentation / oxidation (Melibiose)
AMY: fermentation / oxidation (Amygdalin)
ARA: fermentation / oxidation (Arabinose)
• ONPG test for galactosidase
– an enzyme that cleaves lactose into galactose and
glucose
– organism is grown in a buffered peptone medium
containing D-nitrophenyl-β-D-galactopyranoside
(ONPG)
– production of β-galactosidase is indicated by the
appearance of a yellow color
lactose
– ADH, LDC, and ODC test for enzymes capable of
removing carboxyl groups from the amino acids
arginine, lysine, and ornithine
– Carboxyl group removal is shown by an alkaline
reaction: red color of the particular pH indicator
used)
– Occurs under anaerobic conditions
– CIT tests for the ability of bacteria
to use citric acid
– Broth contains citric acid
– Bacteria with citrate permease
can uptake citric acid, causing
alkaline end products that
change pH indicator to blue
– H2S is produced by some bacteria
when they break down of organic
matter in the absence of oxygen
(anaerobic)
– H2S reacts with the ferrous
sulfate in the media to make
ferrous sulfide…which is black
– presence of a black color
indicates that H2S was
produced.
– URE tests for urease
– Urea, which is usually toxic, is the end product of amino
acid metabolism (anaerobic conditions).
– Some bacteria contain urease, which allows them to break
down urea to form CO2 and ammonia.
– Ammonia production is shown by an alkaline reaction: red
color of the particular pH indicator used)
– TDA tests for tryptophan deaminase
– Tryptophan deaminase removes the amine group
from tryptophan forming indolepyruvate and
ammonia
– demonstrated by the addition of ferric ions
– in the presence of hydrochloric acid the ferric ions
react with the indolepyruvate to produce ferric
hydrazone which is a deep red to brown in color
– IND tests for tryptophanase
– Tryptophanase breaks tryptophan down into
ammonia, indole, and pyrivuc acid
– Kovac’s reagent will react with indole to form a
bright red ring at the surface of the tube
+ H20
NH3 +
+
indole
pyruvic
acid
– VP is used to determine if bacteria can
convert pyruvate to acetoin as part of
the butandiol fermentation pathway
– VP stands for the Volges-Praskauer test
– First alpha-napthol (also called Barritt’s reagent
A) and then potassium hydroxide (also called
Barritt’s reagent B) are added to the VP tube
– The culture should be allowed to sit for about 15
minutes for color development to occur
– if acetoin was produced then the culture turns a
red color (positive result)
– GEL tests for the ability of bacteria to digest the
protein gelatin.
– To digest gelatin, the bacteria must make an enzyme
called gelatinase.
– Bacteria are inoculated onto a solid gelatin tube
which has charcoal at the bottom
– If the bacteria are positive for gelatinase, the gelatin
will liquefy and the charcoal will be distributed
throughout the tube
– GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, and
ARA test for the ability of bacteria to ferment
various carbohydrates (are glucose, mannitol,
inositol, sorbitol, rhamnose, saccharose,
melibiose, amygdalin and arabinose, respectively)
• Bacteria produce acidic products when they ferment
carbohydrates
• Color indicator changes from blue to yellow if acid is
present
Please read through the API20E lab in the lab manual.
The following slides show 3 unknown bacteria that were analyzed
using the API 20E system.
Read and record the result of each strip and determine the API 7digit code for each organism (see next slide for detailed
explanation).
Finally, use the table at the end of the power point to identify
each unknown by Genus species.
Record your results on the API20E Lab answer sheet and upload
the document to the API20E Lab dropbox (you may type your
answers in the textbox if you wish to do so).
API20E Lab Worksheet – finding the 7-digit number
• The test tubes are grouped into 3-tube sets.
• For each tube, mark (+) for positive results and (-) for negative
results as shown above.
• Then give a number value for each tube within each triplet: this
number should be:
• 0 if the test is (-)
• 1 if 1st tube is (+)
• 2 if 2nd tube is (+)
• 4 if 3rd tube is (+)
• Then, add up all three numbers in each set to find the number for
that triplet group.
• Since there are 7 sets, you should have a seven-digit number
API20E Lab Worksheet – finding the 7-digit number
In the example above:
• The 7-digit API20E number is: 5-1-4-7-3-0-6
• Note that the last test, OX, was not performed. A “-“ should
be entered for it.
• Find this number in the reference table or in the online
database to find out which bacterial species it is.
Unknown 1
Unknown 2
Unknown 3
API 20E 7-digit profile
Bacterium
0476021
Proteus vulgaris
1205713
Enterobacter agglomerans
4504552
Salmonella species
4544122
Edwardsiella hoshinae
5144572
Escherichia coli
5215773
Klebsiella pneumoniae
Exercise 8 API 20E Questions.
Name:
Section #
1. Complete the following table for the API 20E strips given in the ppt file:
API ID (7-digit number):
Unknow #
API ID:
Identified Species name
Unknown #1
Unknown #
Unknown #
2. Which group of bacterial species is the API 20E used to identify?
3. Can you use API20E strips to identify Gram positive bacteria? If you try so, what results you might get?
4. Two students perform API20E with the same bacterial culture but get different API numbers. What might be
the reason for this discrepancy?